Nucleic acid amplification is a crucial component of many techniques used in research, medicine, and industry. Such reactions are used in clinical and biological research, detection and monitoring of infectious diseases, detection of mutations, detection of cancer markers, environmental monitoring, genetic identification, detection of pathogens in biodefense applications, and the like, e.g. Schweitzer et al. Current Opinion in Biotechnology, 12:21-27 (2001); Koch, Nature Reviews Drug Discovery, 3:749-761 (2004). In particular, polymerase chain reactions (PCRs) have found applications in all of these areas, including applications for viral and bacterial detection, viral load monitoring, detection of rare and/or difficult-to-culture pathogens, rapid detection of bio-terror threats, detection of minimal residual disease in cancer patients, food pathogen testing, blood supply screening, and the like, e.g. Mackay, Clin. Microbiol. Infect., 10:190-212 (2004); Bernard et al. Clinical Chemistry, 48:1178-1185 (2002). In regard to PCR, key reasons for such widespread use are its speed and ease of use (typically performed within a few hours using standardized kits and relatively simple and low cost instruments), its sensitivity (often a few tens of copies of a target sequence in a sample can be detected), and its robustness (poor quality samples or preserved samples, such as forensic samples or fixed tissue samples are readily analyzed), Strachan and Read, Human Molecular Genetics 2nd Ed. (John Wiley & Sons, New York, 1999).
Because of these advantages, there has been interest in extending amplification techniques to accommodate multiple target polynucleotides from the same biological sample. Several approaches have been taken including (i) simultaneously carrying out multiple amplifications in a single reaction, e.g. multiplex PCR, for example described in Caskey et al. U.S. Pat. No. 5,582,989; Elnifro et al. Clinical Microbiology Reviews, 13:559-570 (2000); Henegariu et al. BioTechniques, 23:504-511 (1997); (ii) sequentially carrying out multiple amplifications in a single reaction by mid-course adjustments in reaction conditions to favor different reactants, e.g. Raja et al. Clinical Chemistry, 48:1329-1337 (2002); and (iii) aliquoting portions of a sample into several reaction chambers for separate amplifications, often using a microfluidics device, e.g. described in Cottingham, U.S. Pat. No. 5,948,673; Mamaro et al. U.S. Pat. No. 6,605,451; Enzelberger et al. U.S. Pat. No. 6,960,437; Liu et al. Anal. Chem., 75:4718-4723 (2003); Woudenberg et al. U.S. Pat. No. 6,126,899; Gulliksen et al. Anal. Chem., 76:9-14 (2004); Anderson et al. U.S. Pat. No. 6,168,948; and the like. The first approach has proven difficult to implement routinely because of the difficulty in finding reaction conditions under which all reactants, such as different primers and target sequences, are amplified at approximately the same rates. The second approach has had some success particularly where rapid amplification of a few sequences of widely varying abundances is required; however, there are severe constraints on available reaction parameters to manipulate in order to obtain preferential amplification of different sequences. The third approach has the potential for permitting multiple amplification reactions to be run on single samples; however, microfluidic devices are generally difficult to manufacture and require complex valving and fluid distribution networks that have impeded their widespread application.
Moreover, in certain applications, such as intraoperation sample testing, and infectious agent and biodefense testing, it is important to employ fluidly closed reaction conditions in order to minimize the occurrence of false positive assessments. The above approaches to analyzing multiple target polynucleotides each introduce level of difficulty either by imposing compromises on the choice of reaction conditions, such that an optimal set of conditions for a reaction as a whole may not be optimal for individual targets, or by requiring access to a reaction after it has been initiated or by employing multiple ports for introducing sample, thereby multiplying chances for contamination, or like problems.
In view of these problems, it would be highly useful for applications requiring rapid amplification of multiple sequences if additional methods were available for such operations that did not have the drawbacks of the current technologies.